Analytic substrate coating apparatus and method

ABSTRACT

An apparatus and method for producing a coated analytic substrate using a compact and portable automated instrument located in the laboratory setting at the point of use which can consistently produce one or a plurality of coated analytic substrates “on demand” for using the analytic substrate immediately after coating, preferably without a step of rinsing the coated analytic substrate before use. The apparatus preferably uses applicator cartridges having a reservoir containing the coating compositions used to form the coatings. Preferably the cartridges are removable and interchangeable to facilitate the production of individual analytic substrates having different coatings or different coating patterns. These coated analytic substrates have superior specimen adhesion characteristics due to the improved quality of the coatings applied by the coating apparatus and due to the quickness with which the coated analytic substrates can be used in the lab after production.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/306,520, filed May 3, 2021, now U.S. Pat. No. 11,307,121, which is acontinuation of U.S. application Ser. No. 16/806,590, filed Mar. 2, 2020now U.S. Pat. No. 10,996,148, which is a continuation of U.S.application Ser. No. 16/291,542, filed Mar. 4, 2019 now U.S. Pat. No.10,578,524, which is a continuation application of U.S. application Ser.No. 15/911,660, filed Mar. 5, 2018 now U.S. Pat. No. 10,222,306, whichis a continuation of U.S. application Ser. No. 15/430,733, filed Feb.13, 2017 now U.S. Pat. No. 9,909,961, which is a continuation of U.S.application Ser. No. 15/018,237, filed Feb. 8, 2016, now U.S. Pat. No.9,568,401; which is a continuation of U.S. application Ser. No.14/473,637, filed Aug. 29, 2014, now U.S. Pat. No. 9,255,863; which is acontinuation of U.S. application Ser. No. 13/220,424, filed Aug. 29,2011, now U.S. Pat. No. 8,820,378; which is a continuation of U.S.application Ser. No. 11/404,702, filed Apr. 14, 2006, now U.S. Pat. No.8,006,638; which claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Application Ser. No. 60/671,746, filed Apr. 15, 2005, theentireties of each of which being hereby expressly incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Methods of coating an analytic substrate, (also referred to herein forexample as a microscope slide or analytic plate), with chemicalcompositions that enhance the adhesion of biological specimens (e.g.,specimens such as cells, tissues, fluids, biological micro-molecules andmacro-molecules) to the analytic substrate are well known. Originally,analytic substrates were often coated by dipping them in commonproteinaceous materials like gelatin or albumin. These compositionswould provide the analytic substrate with a weak adhesive property foradhering the biological specimen to the analytic substrate. However,because there was a need for an enhanced adhesive effect, the process ofcoating analytic substrates evolved to use other procedures andmaterials, for example utilizing polymers of positively charged aminoacids such as L-lysine (i.e., poly L-lysine). In this method, theanalytic substrate was dipped into a 2 to 5 percent solution of polyL-lysine dissolved in a common laboratory solvent such as an alcohol oracetone. These methods produced analytic substrates having improvedadhesive properties, but they proved to be inadequate when used withnewly developed procedures in the laboratory which subjected theanalytic substrate, with the biological specimen attached thereto, toextremely harsh environments or conditions such as enzymatic digestion,microwave boiling, pressure cooker treatments, steamer boiling, andin-situ hybridization protocols.

The composition most often used today for making positively coatedanalytic substrates which are able to withstand the most demandinglaboratory procedures is the silicone polymer3-aminopropyltriethoxysilane. The term “positively charged analyticsubstrate”, as used herein, relates to the “positive” electrostaticcharge the coating imparts on the glass surface. This “positive” netcharge of the coating attracts the typically “negative” net charge ofthe biological specimen.

The method commonly used today for producing a positively chargedanalytic substrate (also referred to herein as a “coated analyticsubstrate”) in a laboratory setting is to dip the analytic substrate ina 2% percent solution of 3-aminopropyltriethoxysilane in acetone for 2to 10 minutes. The analytic substrate thus treated is then rinsed ineither several changes of deionized water or fresh acetone and is thenair dried at room temperature, or heated for example at 60° C. for 60minutes, or overnight. The coated analytic substrates are then stored ina dust free container at room temperature until used in the lab.

To carry out this procedure, laboratory personnel must spend significantamounts of time unpacking plain “untreated analytic substrates” andplacing them into analytic substrate racks (e.g., microscope slideracks) which separate individual analytic substrates from each other sothat all surfaces of each analytic substrate can be coated. Thiscumbersome manipulative procedure of taking each individual analyticsubstrate from its original packing, placing it in a rack, dipping itinto the coating solution, rinsing it several times, then drying, andrepacking the analytic substrates for storage has proven not to be costeffective for most laboratories. Further, the quality of the coating onthe analytic substrate produced in this way varies due to knownvariables which are uncontrollable, and unknown variables that ariseduring the process, such as differences in concentrations among batchesof purchased coating compositions, differences in concentrations ofbatches of working coating composition solutions prepared by differentpersonal, differences in the types of mixing equipment used (i.e.,pipettes vs. graduated cylinders), calibration variation, temperaturechanges, and degradation of the working coating solution during use.

Furthermore, when time protocols are not strictly followed during thecoating process, several inconsistencies in the charged coating on theanalytic substrate can result among batches of coated analyticsubstrates. For example, an “undercoated” or “undercharged” analyticsubstrate results in poor adhesion of the specimen to the analyticsubstrate thereby leading to detachment of the specimen from theanalytic substrate, an obviously undesirable occurrence. “Overcharging”or “overcoating” the analytic substrate by increasing the concentrationof the coating solution applied to the analytic substrate, or byincreasing the time of treatment, often renders the analytic substrateextremely hydrophobic (“low wettability”) such that the analyticsubstrates exhibit decreased or unimproved adhesion characteristics andmay cause non-specific attachment to the analytic substrate of testingchemicals (e.g., dyes and pigments) used for the visualization of thespecimen. An excessively hydrophobic condition is detrimental toautomated staining instruments that require the analytic substrate tomaintain its wettability since as the positive charge of the analyticsubstrate increases, so does the hydrophobicity (liquid repellency) ofthe analytic substrate.

These inherent problems in quality consistency of the analytic substratecoatings are due to a high degree of human involvement necessary toproduce these coated analytic substrates. Therefore, since there is anoverwhelming demand by technicians for these types of positively chargedcoated analytic substrates on a daily basis, laboratories have opted topurchase quantities of ready-to-use charged analytic substrates fromcommercial laboratory supply companies. Unfortunately, all of theproblems and inconsistencies in quality are still experienced in thesecommercially available analytic substrates even after the best effortsof assembly line production of coated analytic substrates from thesevendors.

For example, the dipping process used by manufacturers generallyproduces an uneven coating on the surface because after the analyticsubstrate is dipped into the working concentration of the coating, thecoating material on the analytic substrate is diluted during the rinsesteps thereby often producing an uneven coating. Uneven dryingconditions further produce the uneven coating. Even with increasedconcentration and/or increased time, there is a diminishing return sincethe ability of the coating composition to bind to the glass surface ispartially based on the physical limitations of the chemical entity beingin intimate contact with the glass surface. Furthermore, it is known inthe art that the prior art process of dipping the analytic substratesinto the coating composition produces a analytic substrate that iscoated on all surfaces of the analytic substrate. This is wasteful,because only one analytic substrate surface, herein referred to as the“functional side,” “specimen side,” “upper side,” or “upper surface”needs to be coated for use.

These inconsistencies in quality cause discouragement and frustration inthe laboratory personnel who purchase these ready-to-use coated analyticsubstrates. There is therefore a worldwide consensus regarding the needfor an improved coated analytic substrate that imparts adhesionproperties which are superior to the current laboratory coated orcommercially coated analytic substrates, but most importantly which isreproducible and consistent in quality and function.

SUMMARY OF THE INVENTION

The present invention is an apparatus and method for producing a coatedanalytic substrate using a compact and portable automated instrumentlocated in the laboratory setting at the point of use which canconsistently produce one or a plurality of coated analytic substrates“on demand” for using the analytic substrate immediately after coating,preferably without a step of rinsing the coated analytic substratebefore use. After being produced, the production of these coatedanalytic substrates, due to on-site automation, is extremely costeffective in comparison to the coated analytic substrates known in theprior art. The apparatus preferably uses cartridges having a reservoircontaining the coating compositions used to form the coatings.Preferably the cartridges are removable and interchangeable tofacilitate the production of individual analytic substrates havingdifferent coatings or different coating patterns.

These coated microscope analytic substrates have superior specimenadhesion characteristics due to the improved consistencies inherent incoating apparatus and due to the quickness with which the analyticsubstrates can be used in the lab after being coated.

The present application may contain subject matter in common with U.S.Pat. Nos. 5,948,685; 6,372,006; 6,818,451; 6,555,384; 6,713,304; andU.S. patent application Ser. Nos. 10/944,522; 10/805,777; 10/989,785;and 10/990,080, each of which is expressly incorporated by referenceherein its entirety.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side view of an apparatus of the present invention.

FIG. 2 is a top plan view of an apparatus of the present invention.

FIG. 3 is a top plan view of an alternate version of the apparatus.

FIG. 4 is a cross-sectional view of an applicator cartridge of thepresent invention.

FIG. 5 is a bottom plan view of the cartridge of FIG. 4 .

FIG. 6 is a cross-sectional view of an alternate version of anapplicator cartridge of the present invention.

FIG. 7 is a bottom plan view of the cartridge of FIG. 6 .

FIG. 8 is a cross-sectional view of an alternate version of anapplicator cartridge of the present invention.

FIG. 9 is a bottom plan view of the cartridge of FIG. 8 .

FIG. 10 is a cross-sectional view of an alternate version of anapplicator cartridge of the present invention.

FIG. 11 is a cross-sectional view of an alternate version of anapplicator cartridge of the present invention.

FIG. 12 is a side view of an application method using an apparatus ofthe present invention.

FIG. 13 is a side view of an alternate application method using anapparatus of the present invention.

FIG. 14 is a side view of an alternate application method using anapparatus of the present invention.

FIG. 15 is a side view of an alternate application method using anapparatus of the present invention.

FIG. 16 is a side view of an alternate application method using anapparatus of the present invention.

FIG. 17 is a top plan view of a coated analytic substrate of the presentinvention.

FIG. 18 is a top plan view of an alternate version of a coated analyticsubstrate of the present invention.

FIG. 19 is a top plan view of an alternate version of a coated analyticsubstrate of the present invention.

FIG. 20 is a top plan view of an alternate version of a coated analyticsubstrate of the present invention.

FIG. 21 is a top plan view of an alternate version of a coated analyticsubstrate of the present invention.

FIG. 22 is a top plan view of an alternate version of a coated analyticsubstrate of the present invention.

FIG. 23 is a side view of another embodiment of an applicator cartridgeof the present invention.

FIG. 24 is a side view of the applicator cartridge of FIG. 23 whenactivated.

FIG. 25 is a side view of another embodiment of an applicator cartridgeof the present invention.

FIG. 26 is a side view of another embodiment of an applicator cartridgeof the present invention.

FIG. 27 is a side view of another embodiment of an applicator cartridgeof the present invention.

FIG. 28 is a side view of another embodiment of an applicator cartridgeof the present invention.

FIG. 29 is a top plan view of a movable applicator cartridge holderwhich holds more than one applicator cartridge.

DETAILED DESCRIPTION

As noted above, there has been a long-sought need for coated analyticsubstrates (e.g., coated microscope slides) which have consistentadhesive properties which are sufficient to strongly adhere a biologicalspecimen to the analytic substrate. Methods known in the art of coatinganalytic substrates for the attachment of biological specimens call forthe analytic substrates to dipped and do not have the ability to depositan even and precise amount of the coating onto the analytic substrates.These methods produce analytic substrates which are typically stored forweeks, months or even years before use during which time they candegrade further. The present invention provides a method and apparatusfor producing analytic substrates which overcome the shortcomings ofprior art methods.

In one embodiment, the present invention is directed to a portableapparatus and method for rapid “on demand” production of coatedlaboratory analytic substrates used in the testing of laboratoryspecimens. The term “analytic substrate” where used herein refers to,but is not limited to, analytic plates, microscope plates, andmicroscope slides and is described in greater detail below. The analyticsubstrates may have positively charged coatings, hydrophobic coatings(liquid and specimen retaining borders), anionic, cationic, and/orneutral coatings provided at the point of use, e.g., in a laboratory.

The analytic substrates may be constructed of glass, plastic, syntheticpolymers, or ceramics, and may be of any size or shape known in the artof laboratory examination, for example including any laboratory supportstructure or testing structure or device used in laboratory testing orexamination including, but not limited to, microscope analytic plates,microscope slides, test tubes, Petri dishes, micro arrays, biochips,testing plates, containers, beads, and testing strips. The coating onthe analytic substrate is preferably stable for at least several daysand is preferably activated by hydrolysis from the water contained in abiological specimen around the biological specimen (e.g., buffers ordiluents or other aqueous materials in contact with the specimen) orwater from a water bath for floating biological tissue sections ontoanalytic substrates when the specimen is applied to the analyticsubstrate. Without wishing to be held to theory, it is thought that thewater hydrolyzes the coating composition (e.g., a silane) which thenlinks to the analytic substrate surface and which allows formation ofother linkages between the analytic substrate and the biologicalspecimen disposed thereon, thereby increasing adherence of thebiological specimen to the analytic substrate. The resulting analyticsubstrate is preferably wettable and not overly hydrophobic (except foralternate coating types which impart an excessively hydrophobic surfaceto the analytic substrate for the particular desired results such as forexample hydrophobic borders described in U.S. Pat. No. 5,948,685) thusfacilitating the further treatment of the biological specimen with fluidtreatments that require the analytic substrate to be wettable.

Where used herein the term “biological specimen” includes, but is notlimited to, unprocessed specimens, processed specimens, paraffinembedded tissue, whole mounts, frozen sections, cell preps, cellsuspensions, touch preps, thin preps, cytospins, and other biologicalmaterials or molecules including blood, urine, cerebrospinal fluids,pleural fluids, ascites fluids, biopsy materials, fine needle aspirates,pap smears, swabbed cells or tissues, microbiological preps includingbacteria, viruses, parasites, protozoans, proteins, DNA, RNA,carbohydrates, lipids, microarrays, ELISA reagents and analytes,synthetic macromolecules, phospholipids, support structures ofbiological molecules (e.g., metals, beads, plastics, polymers, glass),or any other materials attached to a biological testing substrate forprocessing, examination, or observation.

Positively coated analytic substrates of the prior art are typicallycoated using silane molecules with a central silicon (S) atom bound tosilyl groups (OR) and functional groups (Y) having the generalstructure:

The functional groups (Y) on the primer or coupling agent (i.e., silanesor siloxanes) in one embodiment of the present invention link thebiological specimen to the analytic plate, and in other embodiments mayfunction in an alternate manner so as to have hydrophobic propertieswherein the primer or coupling agents function as hydrophobic coating onthe analytic plate or function to form a hydrophobic border on theanalytic plate. These and other properties of the primer and couplingagents, which may be related to their use on an analytic plate,including their hydrophobic properties, hydrophilic properties, adhesiveproperties and ionic properties (cationic, anionic, neutral) are furtherdescribed in the Encyclopedia of Polymer Science and Technology, SecondEdition, John Wiley and Sons, 2005 which is expressly incorporated byreference herein in its entirety.

Without wishing to be held to theory, it is known in the art of siliconeattachment to glass that there is generally initially a hydrolysis step(silanol formation) in which the coated analytic substrate is exposed towater (thereby hydrolyzing the silane), and a condensation step whereinthe silanol compound binds to OH groups on the glass surface to formsiloxane bonds (Si—O—Si) between the silicon and the glass surface andproviding exposed Y groups on the glass surface. Also, Van der Waal'sforces, hydrogen bonding and covalent bonding are possible throughsilanol groups.

Hydrolysis:

Condensation (Coupling):

The hydrolysis step could be eliminated in the laboratory or commercialproduction if the coated analytic substrates are only rinsed in acetoneor other non-aqueous solvents prior to drying the analytic substrates.Even though some procedures rinse the coated analytic substrates inwater prior to drying, the glass surface is not effectively coated viacondensation of the silanol composition because dipping is aninefficient way of applying the silicone composition to the analyticsubstrate.

All the previously known positively-coated coated analytic substratesproduced in labs or in commercial settings are produced using some typeof a rinse step after the coating is applied, either in the solvent thecoating material was dissolved in or in a water rinse prior to drying.All these procedures produce a coated analytic substrate that hascompleted its hydrolysis step, if at all, and is dried and packagedprior to use in the lab wherein the specimen is disposed on the analyticsubstrate. Analytic substrates which are produced in this manner beginto lose their functional properties (adhesiveness) in storage forexample due to contamination by environmental contaminates (i.e., dust,humidity) thereby rendering the functional groups on the analyticsubstrate surface ineffective in binding to the specimen.

The primary disadvantage of the laboratory of commercially-produced orlaboratory-produced coated analytic substrates is that the hydrolysisand condensation step has been completed well in advance (e.g., weeks,months or even years) of their use in the lab for attachment of thebiological specimen. This preapplied coating is unstable and graduallyloses its adhesiveness and consistency of adhesion over time duringstorage.

In the present invention, a coated analytic substrate is automaticallyproduced which is preferably used immediately or within a matter ofminutes, hours or days and wherein the hydrolysis and condensation stepsof the coating do not occur until that time when the specimen is appliedto the glass surface (due to the interaction of the water contained onor within the biological specimen, aqueous specimen preparation,buffers, diluents, other solutions, or the histologic water bath). Thisin-situ hydrolysis and condensation, which occurs within the freshlyapplied coating, binds the specimen to the glass in one step therebyproviding superior adhesion of the specimen to the coated analyticsubstrate at the exact time the specimen is applied to the coatedanalytic substrate. As used herein, the term “automatic” refers to anapparatus which may be powered by AC or DC current (includingbatteries), by solar power, by hand-cranking or foot cranking or otherpower-driven means.

Preferably the analytic substrate of the present invention is coatedwith a coating composition comprising one or more silicon-basedcompounds, including, but not limited to silicones, silicon fluids,silanes or siloxanes. Silanes that may be used in the present inventioninclude, but are not limited to: vinylsilanes including:vinyltrichlorosilane, β-methoxyethoxy silane, vinyltriethoxysilane,vinyltrimethoxysilane; acryloxy silanes including:3-metacryloxypropyl-trimethoxysilane; epoxysilanes including: β-3,4epoxycyclohexyl)-ethyltrimethoxysilane,r-glycidoxypropyl-trimethoxysilane,r-glycidoxypropyl-methylidiethoxysilane; aminosilanes including:N-β(aminoethyl)-r-aminopropyl-trimethoxysi lane,N-β(aminoethyl)-r-aminopropyl-methyldimethoxysilane,3-aminopropyltriethoxysilane, N-phenyl-r-aminopropyl-trimethoxysilane;mercaptosilanes including: r-mercaptopropyl-trimethoxysilane,3-mercaptopropylmethyldimethoxy silane; isocyanotosilanes including:3-isocyanatopropyltriethoxysilane; and chloropropylsilanes including:r-chloropropyl-trimethoxysilane. Other silicon-based compositions whichcan be used include styrylsilanes, fluorosilanes, siloxanes,polysiloxanes, crosslinkers, mixed silanes, and other organosilanes,silane coupling agents chrome complexes and titanates, and polymers ofthe chemicals described herein, and combinations of any of the coatingchemicals described herein. The percentage of the silane or siloxanepreferably ranges from less than 0.0001% to in excess of 80% of thecoating composition. The Encyclopedia of Polymer Science and Technology,Second Edition, 2005 includes examples of these and other silanes whichmay be used in the present invention.

In the present invention, the apparatus and method preferably includes astep of physically wiping the coating on the surface using physicalpressure wherein the coating composition is substantially evenly loadedonto the surface of the analytic substrate. This wiping step includesapplying physical pressure of the coating upon the glass therebyphysically embedding the coating into the microscopic ridges, valleys,and pores of the glass thereby enhancing the ability of the coatingcomposition to bind to the surface.

As noted above, even though some procedures known in the art include astep of rinsing the coated analytic substrates in water prior to drying,by doing so, the glass surface is not effectively coated viacondensation of the silanol groups because dipping is an inefficient wayof applying the silicone composition to the analytic substrate. When thecoated analytic substrate is moved from the non-aqueous solvent (e.g.,acetone) to the water rinse, only a minimal amount of coating remains incontact with the analytic substrate (simple surface tension of thecoating to the analytic substrate surfaces). This contact of the coatingis not in the form of siloxane bonds because there has not been adequatehydrolysis of the coating such that it can form silanol groups that canin turn form siloxane bonds with the analytic substrate surface.

Without wishing to be held to theory, it is presumed that when theanalytic substrates are dipped into a water rinse the hydrolysisimmediately begins. This violent mixing of a non-aqueous solvent in thewater rinse apparently inherently causes micro stirring of the coatingcomposition upon the analytic substrate surface. The movement of thesolvents to become miscible to the point of equilibrium producesmicro-currents around the analytic substrate. These micro currentscontribute to the loss of intimate contact of the silanol groups neededto form siloxane bonds to the analytic substrate surface which isnecessary for the coating material to link to the analytic substratesurface. Although one might conjecture that any coating material thatwas transferred from the coating composition to the water rinse wouldbecome hydrolyzed and would be available to bind to the analyticsubstrate surface, it is anticipated that the inherent problem is theintimate area around the analytic substrate would have extremely dilutedhydrolyzed coating composition around the analytic substrate which wouldbe available to bind to the analytic substrate surface. It is alsoapparent that when analytic substrates are dipped by moving the analyticsubstrates up and down in the water rinse or causing a stirring actionin the water rinse container, further loss of the coating material awayfrom the analytic substrate surfaces occurs and/or a patchy distributionof the coating occurs thereon. These prior art methods result in poorlycoated analytic substrates with uneven coatings and weak adhesiveproperties. It is also known that this type of method employs multiplerinse steps which obviously would produce an even lesser coated analyticsubstrate with poorer adhesive properties.

In the present invention, the coating composition is already in intimatecontact with the analytic substrate surface due to physical embedding ofthe coating by the applicator. Further intimate contact with the silanolgroups and the analytic substrate surface during hydrolysis is realizedin the present invention, when the water from the biological specimen orwater contained in aqueous processing buffers or the histologic waterbath is trapped under the specimen providing a localized hydrolytic areatherebeneath by leaving minimal space for the silanol groups to bedispersed away from the analytic substrate surface. The biologicalspecimen itself traps the water and produces an intimate and localizedhydrolytic area under itself were the silanol groups can concentrate andfully condensate to the analytic substrate surface. The efficientcondensation of the coating which occurs during use of the presentinvention produces an increased formation of siloxane bonds over thecoated analytic substrates of the prior art, which results in anincreased adhesion of the biological specimen due to increaseconcentrations of siloxane bond formation. In summary, this intimate andlocalized hydrolytic area along with the biological specimen, primer orcoupling agent (e.g., silane, siloxane), and glass surface producesmicro-environment for a superior link of siloxane bonds to the analyticsubstrate surface and superior link of the functional group of theprimer or coupling agent to the biological specimen.

Solvents which can be used in the coating composition of the presentinvention include, but are not limited to, alcohols, including methyl,ethyl and isopropyl alcohol, acetone, ketones, MEK, xylene, toluene,benzene, and aqueous and non-aqueous and/or organic and inorganicsolvents, including polar and non-polar solvents. The solvent typicallycomprises from 20% to 99.9999% of the coating composition. The coatingcomposition may further comprise a surfactant (anionic, cationic, orneutral). The coating composition can have a pH that favors cationic,anionic, neutral, hydrophobic, and hydrophilic coatings. Thus, the pH ofthe coating composition can be in the range of 1-10 depending on thedesired electrostatic charge intended for a particular coating.Preferably the coating is in the acidic range to impart a cationiccharge to facilitate its electrostatic binding to the biologicalspecimen. There are numerous chemicals known that can be used to alterthe pH of the coating composition, including, but not limited to aceticacid, benzoic acid, ethyl sulfate, potassium hydroxide, sodiumhydroxide, other organic and inorganic acids and bases.

In alternative embodiments, the coating composition may include an acidcatalyst, a base catalyst, or other catalysts known in the art orcombinations thereof which increase the rate of pre-hydrolysis andpre-condensation or complete condensation of the silane in the coatingcomposition. This catalyst results in complete or partial hydrolysisand/or complete or partial condensation of the coating composition whenthe coating composition is applied to the analytic substrate, before thebiological specimen is applied to the analytic substrate. The catalystwhen present may comprise, for example, from less than 0.00001% to 20%of the coating composition.

Thus the hydrolysis step and condensation step can occur without thesimultaneous application of the biological specimen. Analytic substratesproduced in this manner can be stored for later application of thebiological specimen to the analytic substrate.

In summary, in a preferred embodiment of the invention, the coatingcomposition is applied to the analytic substrate, which is free of abiological specimen disposed thereon, then the biological specimen isquickly applied to the analytic substrate. Water which initiates thehydrolysis step is provided by the water contained on or within thebiological specimen, aqueous specimen preparation, buffers, diluents,other solutions, or the histologic water bath.

As noted, alternatively, the coating composition can include a catalyst(e.g., an acid or base). In this embodiment when the coating compositionis applied to the analytic substrate, the hydrolysis and condensationsteps occur completely, or at least partially, before the biologicalspecimen is applied to the analytic substrate. When the biologicalspecimen is applied some further hydrolysis might occur.

Alternatively, the coating composition may comprise water (from lessthan 0.0001% to 99%), wherein when the coating composition undergoes atleast partial hydrolysis and condensation on the analytic substratebefore the biological specimen is applied to the analytic substrate.Further hydrolysis and condensation may occur when the biologicalspecimen is applied to the analytic substrate.

Finally, the coating composition may comprise both a catalyst and waterwherein at least partial hydrolysis and condensation occurs on theanalytic substrate before the biological specimen is applied to theanalytic substrate. When the biological specimen is applied some furtherhydrolysis might occur. Examples of catalysts which may be used include,but are not limited to, organic and inorganic acids and bases including:amines, benzoic acids, KOH, acetic acid, n-propylamine, metals, andphenyl-β-naphthylamine. Further benefits by the present invention by thepre-hydrolysis containing coatings are explained. The pre-hydrolyzedcoating embodiments would cause the attachment and immobilization of theprimer or coupling agent to the analytic substrate by at least onesiloxane bond. This advantage of pre-condensation of the primer orcoupling agent to the analytic substrate increases the amount ofsiloxane bonds because the primer or coupling agent is already attachedto the analytic substrate and cannot be diluted or washed away. Onlyfurther hydrolysis and condensation can occur with the addition of thebiological specimen. This benefit is realized with coatings of thepresent invention that provide pre-hydrolysis with or without a catalystbefore the addition of the biological specimen. The prior art methods donot teach pre-hydrolysis solutions with or without a catalyst.

The present invention benefits in some embodiments from the release ofalcohol during the in-situ hydrolysis of some silanes. The presence ofalcohol is advantageous because it will flatten out or level paraffinembedded tissue sections (biological specimens) by removing residualwater trapped underneath the section from the histologic floatationwater bath or by removing water inherent in the biological section(e.g., in frozen sections) thereby decreasing the drying time necessarybefore further processing. This production of alcohol can beadvantageous when attaching frozen sections (fresh tissue) to ananalytic plate. It is common in the art to place a frozen sectionmounted on a microscope analytic plate in 95% alcohol immediately afteradherence to the analytic plate for fixation prior to staining. Thein-situ production of alcohol, from hydrolysis of the coated analyticplate, penetrates through the frozen biological specimen (i.e., tissue)at the time of attachment of the frozen section to the coated analyticplate and starts the fixation process immediately. Since the coating isbeing hydrolyzed by the water from the fresh frozen biological sectionthe production of alcohol during the condensation step could speed upthe time for fixation in a separate container of alcohol or couldeliminate altogether this step of fixation in 95% alcohol, therefore,the technician could go straight from mounting the frozen section to thestaining process and same time by eliminating the step of fixation in acontainer of 95% alcohol prior to staining.

In the present invention, the coating is applied only to the uppersurface (functional side) of an analytic substrate and is, inparticular, applied to at least a portion of the functional side of thesubstrate upon which the biological specimen will be positioned anddisposed. In an alternate embodiment, both surfaces (upper and lower) ofan analytic substrate can be coated either separately or simultaneously.The coating can cover the entire analytic substrate even in areas whereno biological specimen would be located. This embodiment of completelycoating an entire surface area of an analytic substrate (i.e., theentire upper surface of a microscope analytic substrate) not pertinentto attaching to the biological specimen or the function of the borderedareas around the biological specimen could help in the adhesion of inksand writing device markings (e.g., ink pens) to identify the analyticsubstrate. It is known in the art that silanes can help bond inks toglass, plastic, and ceramic surfaces. There is usually an area on ananalytic substrate (i.e., a frosted end, unfrosted end and/or an opaquepainted end of a microscope slide) where identification markings areplaced on the analytic substrate. The coating contemplated herein couldhelp in the adherence of these markings by reacting with the markingchemicals and the silane functional groups attached to the analyticsubstrate, for example, thus providing a stronger bond of the markingchemicals to the analytic substrate by the coatings functional groupsstrongly attached to the analytic substrate. As noted above, the coatingpreferably is a silicone, silicone fluid, silane, fluorosilane,organofunctional silane, siloxane, polysiloxane, and/or any combinationsof the above or any polymers of silicone or other coating compositionthat binds to a surface of an analytic substrate and which forms a linkto the surface of the analytic substrate and has at least one functionalgroup that can bind to a biological specimen and at least one reactivesilyl group (and preferably three) which binds to the analyticsubstrate. Thus, the coating composition, once linked to the analyticsubstrate, would have functional group properties for adheringly bindingthe specimen to the plate. This process can also be used on plasticplates as noted herein.

The invention contemplates a self-contained compact, portable, automatedinstrument that can be located in a laboratory setting for producingindividually coated analytic substrates having a coating compositionapplied by automatically wiping the coating composition onto the uppersurface of the analytic substrate with an applicator device (applicatorcartridge) having an applicator device (also referred to herein as anapplicator end). The coating composition can be wiped, sprayed, gravurecoated, reverse roll coated, roll coated, gap coated, Meyer rod coated,slot die coated, curtain coated, or air knife coated upon the analyticsubstrate. The invention contemplates automatically applying the coatingcomposition to a single analytic substrate, a plurality of analyticsubstrates at the same time, or a plurality of analytic substratescoated one at a time.

A particular advantage of the present invention is that the replaceable,interchangeable applicator cartridges of the apparatus allow the user toquickly and easily change the coating compositions and formulations usedin the apparatus. For example the user can use one applicator cartridgeto produce a few analytic substrates for a particular type of biologicalspecimen, then can replace it with a different applicator cartridgehaving a reservoir containing a different coating composition to producea few analytic substrates for use with an altogether differentbiological specimen. It is well known in the art that certain types ofbiological specimens (e.g., paraffin embedded tissues such as braintissue, breast tissue, fatty tissues, keratinized tissue, cartilage,bone, and tissues which are under processed or over processed (e.g.,under-fixed, or over-fixed, respectively)) present greater “analyticsubstrate adherence” problems than other biological specimens. Examplesof problematic unprocessed frozen tissues include, but are not limitedto, breast tissues, fatty tissues, skin tissues and brain tissues. Thepresent invention thus provides the user with a way to “tailor make” ananalytic substrate which can have greater adherence qualities forparticular types of biological specimen. There can be multipleapplicator cartridges present in a single holder to treat the analyticsubstrates with several different types of coatings solutions.

The ability offered by the present apparatus to create “on-demand”specialized types of coated analytic substrates eliminates the need tohold a large inventory of coated analytic substrates in the laboratory,thereby reducing costs and reducing wastage of coated analyticsubstrates due to loss of effectiveness because of short shelf life ofthe coating on the analytic substrates.

In a preferred embodiment an applicator cartridge is used for directapplication and wiping on the analytic substrate wherein the applicatordevice of the applicator cartridge is moistened or pre-moistened withthe coating composition and is pressed upon the analytic substrate, andthe composition is wiped upon the analytic substrate, thereby leaving alayer of coating composition embedded upon the analytic substrate. Theapparatus can be programmed to coat the entire surface of the analyticsubstrate or just portions thereof. This applicator device can wipe theanalytic substrate once or a plurality of times to apply the coatingcomposition to the analytic substrate. Preferably, the applicatorcartridge can also be adjusted to change the amount of pressure beingapplied to the analytic substrate via the applicator device duringwiping. This pressure, for example, can be from 0.01 psi to 0.1 psi to 1psi to over 10 psi (and preferably at least 5 psi) for enhancing theembedding of the coating composition into the analytic substrate.

In one embodiment the coating composition can be sprayed on the analyticsubstrate and then wiped thereon by the applicator device and then used,without first being rinsed in a rinsing step, or the coating compositioncan be sprayed on the analytic substrate and then used without a rinsingstep. Preferably when the coating composition is sprayed on the analyticsubstrate it is sprayed upon only one surface (the upper surface, i.e.,functional side) of the analytic substrate.

The applicator device can be linked to a coating composition reservoirvia a hose, tube or other conduit (as described elsewhere herein) andthe coating composition can be sprayed onto the applicator devicedirectly thereby moistening the applicator device wherein the applicatorend then wipes the coating composition on the analytic substrate.

The applicator device and reservoir can be a unitary assembly (i.e., anapplicator cartridge) which is disposable and/or reusable. Theapplicator device can be moistened by the coating composition in thereservoir at the time the applicator device is pressed upon the analyticsubstrate. The applicator device can also be a stylus, or “stylus-like”.Or the applicator cartridge may have a pen or “pen-like” applicatordevice, or the applicator cartridge may be a pen or “pen-like”.

The reservoir of the applicator cartridge can be pressurized wherebyprior to the applicator device coming in contact with the analyticsubstrate, the coating composition is released to prime and moisten theapplicator end prior to wiping the analytic substrates. The reservoircan be activated by pressure or gravity fed to release the coatingcomposition into the applicator device when it is pressed to theanalytic substrate.

The reservoir can be refillable or disposable (i.e., used one time). Theapplicator cartridge and/or the reservoir can be interchangeable. Thus,an applicator cartridge (or reservoir within the cartridge) can be used,then exchanged with a different applicator cartridge (or reservoirwithin the cartridge), then reused again until the coating compositionis depleted. The applicator device of the applicator cartridge can beconstructed of a material known in the art for such applicators (e.g.,cotton, polyester, polymer, rubber, plastic, silicone, foam, membranetype, perforated metal, absorbent and non-absorbent materials).

This applicator device can be soft, medium, or hard therefore havingdifferent durometer ratings and absorbencies in relation to the degreeto which the applicator device can be wetted with the coating material.The applicator device can be of any size suitable for coating alaboratory plate. The coating material can be a liquid, fluid, gel,semi-solid, or any other consistency known in the art of coatingchemical formulations. The coating composition can be clear,transparent, translucent, non-colored, colored, pigmented,non-pigmented, colloidal, or impart any other physical attributes knownin the art of coating compositions. The coating, once on the analyticsubstrate, can be clear, transparent, translucent, colored, or impartany other physical attributes known in the art once applied to theanalytic substrates. The applicator cartridge may have a compositionwhich has a long shelf life (e.g., several months) after its initialuse, or may have a composition which has a relatively short shelf life(e.g., hours or days) after its initial use. The applicator cartridgemay contain two or more separate compositions which are mixed prior touse of the applicator cartridge or the applicator cartridge may have twoor more separate reservoirs and applicator devices for applying separatecoatings to the analytic substrate.

In a preferred embodiment the analytic substrate is coated only on oneside of the analytic substrate, i.e., the functional side upon which thebiological specimen is to be deposited. Preferably in a furtherembodiment there is no rinse step after the coating process prior to theattachment of the biological specimen. Absence of a rinse step resultsin less dilution of the coating material on the analytic substrate andthus enhance adhesiveness and consistency in thickness of the coating.

In a further embodiment, the coating can be a mono-molecular layer, amulti-molecular layer or a multi-layer in thickness. Since the coatingis preferably applied with an applicator having an applicator device, afirst coating layer can be applied then dried and then a second layercan be applied to form a desired thickness, by applying a multiplenumber of layers. The thickness of the coating can be, for example,1×10⁻¹⁰ m or less, 1×10⁻⁹ m or less, 1×10⁻⁸ m or less, 1×10⁻⁷ m or less,1×10⁻⁶ m or less, 1×10⁻⁵ m or less, 1×10⁻⁴ m or less, or 1×10⁻³ m orless.

In a further embodiment, the coating on the analytic substrate can haveproperties that are hydrophobic, hydrophilic, neutral, cationic andanionic. The apparatus can separately deposit several different types ofcoatings (e.g., hydrophobic, hydrophilic, adhesive) on one analyticsubstrate. In a preferred embodiment, the instrument can automaticallyapply an adhesive coating to the surface of the analytic substrate as afirst coating, allow the analytic substrate to dry and then apply ahydrophobic second coating to selective areas on the analytic substrate(e.g., as a containment border) in any pattern desirable to containfluids or biological specimens to specific portions of the analyticsubstrate. Thus the prepared analytic substrate could have both adhesiveareas and hydrophobic areas present on the analytic substrate. Anycombination of coatings can be applied by the instrument to the analyticsubstrate. Any pattern of any type of coating can be applied to theanalytic substrate. The instrument can apply a single coating to theentire functional side of the analytic substrate or just selective areason the analytic substrate. Thus, the instrument may have multipleapplicator cartridges (and or multiple applicator ends), and multipledifferent coating compositions, to coat the analytic substrate with anypattern. The applicator devices of the applicator cartridges can besized to cover the entire surface area of the analytic substrate withone application or just a portion of the surface area. The applicatordevices can be stylus-like or pen-like to apply the coating material tospecific areas to the analytic substrate. Preferably the adhesivecoating is invisible once mounted with a coverslip to allow light (i.e.,transmitted, fluorescent) to pass through the coating without changingthe refractive index of the glass. In an alternate embodiment of theadhesive coating, the coating once mounted with a coverslip istransparent and non-colored, or transparent and colored. The hydrophobicborder coatings (where present) may be invisible, transparent andnon-colored, colored and transparent, translucent and colored, andtranslucent and non-colored. The coating in a preferred embodiment doesnot change the refractive index of the analytic plate when viewedtherethrough.

The preferred method of applying the coating material to the analyticsubstrate is by causing intimate physical contact of the coatingmaterial with a surface of the analytic substrate by exerting pressurefrom the applicator upon the coating material on the analytic substrate.For example, the coating material is preferably applied to the analyticsubstrate from the applicator device by means of friction, e.g., wipingor rubbing, or by vibrations, ultrasound, pulsations, or other meansknown in the art.

In a preferred embodiment, once the coating is applied to the analyticsubstrate, the analytic substrate is ready for use. The analyticsubstrate can be used immediately, or within seconds, minutes, hours,days, weeks, or even longer after being coated and still be effective inits use based on the coating material used and the intended use (i.e.,hydrophobic properties, hydrophilic properties, adhesive properties,neutral, cationic, anionic, etc.). Preferably the analytic substrate isused within seconds or minutes after coating.

Referring now to the drawings, preferred embodiments of the apparatus ofthe invention are shown in FIGS. 1-3 . FIGS. 1 and 2 show an analyticsubstrate coating apparatus 10. The analytic substrate coating apparatus10 has an applicator cartridge holder 11 which has an armature 12 forsupporting the applicator cartridge holder 11 on an X-Y-Z positioningmechanism which comprises a vertical movement post 16. The applicatorcartridge holder 11 supports a removable applicator cartridge 14 whichhas an applicator device 15 as described in more detail below andelsewhere herein. The armature 12 is connected to the vertical movementpost 16 via a vertical drive motor 18 (or to other drive means as knownin the art) which raises and lowers the applicator cartridge holder 11and applicator cartridge 14. The analytic substrate coating apparatus 10also has a lateral drive rail 20 having a lateral drive motor 22 (orother drive means as known in the art) disposed thereon which isattached to the vertical movement post 16 and is moved laterally by thelateral drive motor. The analytic substrate coating apparatus 10 furtherhas a left forward/reverse drive rail 24 and a right forward/reversedrive rail 26. The lateral drive rail 20 is connected to the leftforward/reverse drive rail 24 via a left drive motor 28 (or other drivemeans as known in the art) and the right forward/reverse drive rail 26is connected to the lateral drive rail 20 via a right drive motor 30 (orother drive means as known in the art). The left drive motor 28 andright drive motor 30 drive the lateral drive rail 20, and thus theapplicator cartridge 14 in a forward direction and reverse directionduring use. In an alternate embodiment of the invention, the analyticsubstrate coating apparatus 10 may be constructed with only a singleforward/reverse drive rail (not shown). An alternate example of an X-Y-Zpositioning mechanism and a control mechanism for operating it such asmay be used in the present invention is shown in U.S. Pat. No. 5,443,791the entirety of which is hereby expressly incorporated herein byreference. The analytic substrate coating apparatus 10 optionally has ananalytic substrate supply bin 36 (also referred herein as an analyticsubstrate hopper or analytic substrate supply station) which isconstructed to hold an analytic substrate stack 38 comprising aplurality of analytic substrates 40 and which further has a analyticsubstrate ejection mechanism 42 for ejecting or pushing a singleanalytic substrate 40 onto a analytic substrate support mechanism suchas a conveyor 44.

The conveyor 44 can be constructed to hold one or more analyticsubstrates 40 for coating and processing as described elsewhere herein.After the analytic substrate 40 has been coated and processed it may bedried and/or cured using a dryer 46 (optional) or other drying or curingmeans before it is delivered to an analytic substrate storage bin (oranalytic substrate receiving station) 48 comprising an analyticsubstrate support 50 and an analytic substrate support mechanism 52 suchas a spring or other similar device known in the art for supportingobjects in a receiving bin (analytic substrate receiving station).

During use of the analytic substrate coating apparatus 10, an analyticsubstrate 40 is removed from the analytic substrate hopper 36 via theanalytic substrate ejection mechanism 42 and is conveyed via theconveyor 44 to a coating position. A coating composition containing theapplicator cartridge 14 is applied to a portion of an upper surface ofthe analytic substrate 40 via a dispenser device of the applicatorcartridge 14. The coating is applied to the analytic substrate 40 in amanner as described elsewhere herein. The analytic substrate coatingapparatus 10 may include an exhaust fan (not shown) with or without anactivated charcoal filter to neutralize any fumes being produced duringthe coating process.

The analytic substrate coating apparatus 10 preferably includes amicroprocessor (not shown, and also referred to herein as a programmablecontrol mechanism) that can be programmed with multiple coatingprotocols which can be selected by the user to produce a particular typeof coated analytic substrate in a manner known to persons of ordinaryskill in the art. Any number of programmable data can be associated withthe apparatus 10 and be either pre-programmed for the user or userprogrammable. The apparatus 10 could even have an electronic pad presentfor the user to draw by hand or by wand (e.g., pen, stylus) a patternwhich is digitized and sent to the microprocessor for the apparatus 10to produce a particular pattern of coating on the analytic substrate 40.The microprocessor may have other abilities known in the art of computercontrollable devices including, but not limited to, a keyboard, akeypad, touch sensitive screen, hard drive, storage devices (e.g.,removal, not removable), and voice recognition.

Preferably, the applicator cartridge 14 can be moved in three dimensions(forward and backward), laterally (side-to-side), and vertically (up anddown) although the applicator cartridge 14 in another embodiment may bemovable only in one or two directions. In a preferred embodiment afterthe analytic substrate 40 is delivered to the preferred coatingposition, the applicator cartridge 14 is lowered on the verticalmovement post 16 to an applicating position wherein the coatingcomposition contained within the applicator cartridge 14 is dispensedupon the analytic substrate 40. When the coating composition is appliedto the upper surface of the analytic substrate 40 it is then preferablywiped or rubbed on the surface of the analytic substrate 40 via means onthe applicator device 15. The coating composition may be applied to theanalytic substrate 40 via a sprayer, roller, sponge, squeegee or otherdevice as discussed elsewhere herein then may be wiped, rubbed or spreadby another device such as a pad or sponge on the applicator device 15 onthe applicator cartridge 14 as discussed elsewhere herein. Theapplicator end 15 may be stationary or mobile. If stationary, theanalytic substrate 40 itself is moved to cause the coating compositionto be wiped or rubbed on the analytic substrate (see FIG. 17 below).After the coating is applied to the analytic substrate 40, it may bedried (e.g., air dried, heat dried, or UV cured) via the drying device46 which may be for example a fan, blower, heating element or UV lightor other such mechanism known in the art. The applicator device 15 mayapply the coating composition to only portions of the analytic substrate40, such as a border which completely or partially surrounds acontainment area on the analytic substrate 40 as described below.

Before going to the analytic substrate storage bin (or analyticsubstrate receiving station) 48, the upper surface of the analyticsubstrate 40 with the coating thereon optionally may be buffed using abuffing device (not shown). Alternatively, a separate hydrophobic bordermay be applied to the analytic substrate 40 after the coating is applied(see FIG. 22 below). Alternatively the separate hydrophobic border maybe applied to the analytic substrate 40 before the coating compositionis applied to the analytic substrate 40. Alternatively, a hydrophobicborder alone may be applied to the analytic substrate 40. During thedispensing of the coating composition upon the analytic substrate 40,the applicator cartridge 14 may be moved laterally on the lateral driverail 20 via the lateral drive motor 22 to apply the coating compositionin a predetermined pattern on the analytic substrate 40, and theapplicator cartridge 14 may also be moved in forward and reversedirections by actuation of the left drive motor 28 and right drive motor30 on the left forward/reverse drive rail 24 and right forward/reversedrive rail 26, respectively, thereby applying the coating composition ina predetermined pattern, or simply to wipe, rub, or spread the coatingcomposition on the analytic substrate to increase its adherence theretoas discussed elsewhere herein.

After the analytic substrate 40 is coated, it is advanced to the dryingdevice 46 (if present) and then to the analytic substrate storage bin 48where multiple analytic substrates 40 can be stored. The applicatorcartridge 14 is then returned to a resting position or an applicationposition where it applies the coating composition to another analyticsubstrate 40.

In another embodiment of the invention, an analytic substrate coatingapparatus 10 a is shown in FIG. 3 . The analytic substrate coatingapparatus 10 a is similar to the analytic substrate coating apparatus 10except it also has a secondary applicator cartridge holder 56 forsupporting a secondary applicator cartridge 58 for applying a secondcoating, marking or pattern onto the analytic substrate 40. Thesecondary applicator cartridge holder 56 is connected to a secondvertical post (not shown) on the lateral drive rail 20 by a secondaryvertical drive motor 60. The second vertical post (not shown) is movedon the lateral drive rail 20 via a secondary lateral drive motor 62.

The applicator cartridge 14 may be constructed in a variety of differentembodiments as shown for example in FIGS. 4-16 and 24-29 . FIGS. 4 and 5show an applicator cartridge 70 having a body 72, a reservoir 74contained within the body 72, and a lower end 76 having a dispenserdevice 78 and a pair of applicator devices 80 (e.g., wiping devices).The reservoir 74 contains a quantity of the coating composition which isdelivered through the dispenser device 78, such as a valve, to an uppersurface of the analytic substrate 40. The applicator devices 80 may be apad or sponge, constructed for example from rubber, a polymeric materialor a fabric such as felt or other absorbent or non-absorbent materialand may be used to spread the coating composition on the analyticsubstrate 40 as discussed elsewhere herein. In a preferred embodiment,the applicator device 80 is constructed of a material and has a sizesuch that when moistened with the coating solution, they retain onlyminimal amounts of coating solution so when drying of the applicatordevice 80 occurs, e.g., when the apparatus is not in use, waste due toevaporation and subsequent loss by the drying of the coating compositionfrom the applicator device 80 is minimized. The applicator device 80, ina preferred embodiment, can be moistened and ready for use with aslittle as 0.01 μl (e.g., from 0.01 μl to 10 μl to 1000 μl to 5000 μl to20,000 μl) of coating composition depending on the analytic substrate(and its size) to be coated. Once the process of coating is started, themicroprocessor will activate the appropriate devices (i.e., pumps,valves, or moistness sensors) to maintain the moistness of theapplicator device 80 to consistently coat each analytic substrate withminimal waste due to drying of the applicator device 80 after theprocess is finished.

FIGS. 6 and 7 show an applicator cartridge 90 as an alternateembodiment. Applicator cartridge 90 has a body 92 which contains areservoir 94 which contains the coating composition. The applicatorcartridge 90 has a lower end 96 comprising a dispenser device 98 whichdispenses the coating composition directly onto a wiper device 100 whichmay be similar in construction to the applicator device 80 of applicatorcartridge 70.

FIGS. 8 and 9 show an applicator cartridge 110 similar to applicatorcartridge 70, having a body 112, a reservoir 114 therein and a lower end116 except the lower end 116 has a dispenser device 118 which is astylus or other marking device, and which does not have an applicatordevice in the lower end 116.

FIG. 10 shows an applicator cartridge 130 which has a body 132, areservoir 134 with a coating composition 136 therein and a lower end 138having a dispenser device 140 such as a squeezable “push up” tube fordelivering a quantity of the coating composition 136 to an applicatordevice 142 such as described previously. For example, when the lower end138 of the applicator cartridge 130 is pressed against the analyticsubstrate 40, the applicator device 142 presses the dispenser device 140which causes the quantity of coating composition 136 to flow into theapplicator device 142 and onto the analytic substrate 40.

FIG. 11 shows an applicator 150 having a body 152 which contains areservoir 154 containing a coating composition 156, and having a lowerend 158 with a dispensing device 160 and an applicator device 162similar to the applicator device 142. The dispenser device 160 is anelectric valve having leads that respond to a pulse of pressure andcause the dispenser device 160 to release the coating composition 156into the applicator device 162.

The coating composition in the applicator cartridge can be applied tothe analytic substrate 40 in a variety of ways. For example, as shown inFIG. 12 , an applicator cartridge 170 having an applicator device 172and which may be any applicator cartridge described herein. Theapplicator device 172 is simply lowered onto the analytic substrate 174and drawn in direction 178 to coat a portion of the analytic substrate174. Shown in FIG. 13 is an applicator cartridge 180 having anapplicator device 182 and a separate dispenser device 184 whichdispenses a quantity of coating composition onto a portion of theanalytic substrate 174 and which is then wiped or spread upon theanalytic substrate 174 when the applicator device 182 is drawn across ofportion of the analytic substrate 174 in direction 178. Shown in FIG. 14is an embodiment of the invention wherein the applicator cartridgedispenses the coating composition directly onto analytic substrate 174via a sprayer dispenser device 184 which moves over the analyticsubstrate 174 in a direction 178. Shown in FIG. 15 is an embodiment ofthe invention wherein an applicator cartridge 180 having applicatordevice 182 has the coating composition sprayed directly thereon from asprayer dispenser device 190 which then applies the coating compositionto the analytic substrate 174 as the applicator device 182 moves overthe analytic substrate 174 in direction 178. Shown in FIG. 16 is anembodiment similar to that shown in FIG. 15 except there are at leasttwo spray dispenser devices 194 which spray the coating composition ontothe applicator device 182 of applicator cartridge 180.

The applicator device 182 (or any other applicator device describedherein) is preferably saturated with the coating composition whenapplied to the analytic substrate 40. Alternatively, if the applicatordevice is dry, it may be primed with the coating composition before theapplicator device is applied to the analytic substrate. The applicatordevice or dispensing device may have a moisture or wetness sensortherein to keep the applicator device or dispensing device moist.

The coating composition can be applied to the analytic substrate to makea variety of patterns useful in the art of histology and medicaltechnology. For example, shown in FIG. 17 is an analytic substrate 200which has an upper surface 202. The upper surface 202 has a largercoated area 204 and a smaller uncoated are 206 (e.g., for use in markingor labeling the analytic substrate 200). Shown in FIG. 18 is an analyticsubstrate 210 which has an upper surface 212 having a first coated area214 and a second coated area 216, and a first uncoated area 218 betweenthe coated areas 214 and 216 and a second coated area 220 for marking orlabeling the analytic substrate 210. Shown in FIG. 19 is an analyticsubstrate 222 having an upper surface 224 and a pair of coated areas 226and 228 which comprise narrow strips on the upper surface 224, which aredisposed between three uncoated areas 230, 232 and 234, of the analyticsubstrate 222. Shown in FIG. 20 is an analytic substrate 236 having anupper surface 238 which has a pair of “circles” 240 and 242 whichcomprise coated areas and an uncoated area 244 on the upper surface 238of the analytic substrate 236. Shown in FIG. 21 is an analytic substrate246 having an upper surface 248, and having a coated area 250 whichcomprises a rectangle (or any other geometric or irregular shape) and anuncoated area 252. Shown in FIG. 22 is an analytic substrate 254 whichhas an upper surface 256 and has a coted area 258 which comprises thecoating composition of the present invention, and a rectangular border262 comprising a hydrophobic composition (such as described in U.S. Pat.No. 5,948,685) which is positioned upon a portion of the coated area258. Analytic substrate 254 also has an uncoated area 260. Any of thecoated analytic substrates described herein may be constructed withoutuncoated area.

Especially preferred embodiments of the applicator cartridge of thepresent invention are shown in FIGS. 23-26 . An applicator cartridge 270shown in FIGS. 23 and 24 comprises a body 272, and a reservoir 274containing a coating composition 276 which comprises, for example, anyof the coating compositions described elsewhere herein. The applicatorcartridge 270 further comprises an applicator device 278 which has slidemeans (not shown) for enabling it to be pushed upwardly into the body272 when the applicator device 278 is pressed against a surface such asan analytic substrate as described elsewhere herein. The applicatorcartridge 270 further comprises a flexible wall 280 positioned withinthe body 272 between the reservoir 274 and the applicator device 278 forcontaining the coating composition 276 within the reservoir 274.

The applicator cartridge 270 further comprises a pumping/dispensingsystem such as, but not limited to, a peristaltic pumping/dispensingsystem 282 within the body 272 and which is positioned partially withinthe reservoir 274 and a space 284 between the flexible wall 280 and theapplicator device 278. The peristaltic pumping/dispensing system 282preferably has at least a single one way valve and more preferably has afirst one way valve 286 and an inlet 288, a second one way valve 290 anda pumping tube 292 operatively connected between the first one way valve286 and the second one way valve 290 for receiving a quantity of thecoating composition 276 from the one way valve 286. A dispensing tube294 having one or more dispensing ports 296 exits the second one wayvalve 290 for delivering the quantity of coating composition 276 fromthe pumping tube 292 onto the applicator device 278 when the peristalticpumping/dispensing system 282 is activated.

The peristaltic pumping/dispensing system 282 is activated when apressure point 298 (e.g., a hump or knob) disposed upon the applicatordevice 278 is pushed upwardly against the flexible wall 280 when theapplicator cartridge 270 is pushed downwardly against an analyticsubstrate. When the pressure point 298 presses against the flexible wall280, the flexible wall 280 is in turn pressed against the pumping tube292 which in turn is pressed against a rigid plate 300 (as shown in FIG.24 ) which thereby compresses the pumping tube 292, thereby forcingcoating composition therein through the second one way valve 290 intothe dispensing tube 294 and therefrom through the dispensing ports 296and into the applicator device 278, which then spreads the coatingcomposition 276 onto the analytic substrate as described elsewhereherein. When pressure from the pressure point 298 is released, thepumping tube 292 is decompressed which causes a new portion of coatingcomposition 276 to be drawn through the inlet 288 into the first one wayvalve 286 and therefrom into the pumping tube 292. The process can thenbe repeated.

In an alternate embodiment, an applicator cartridge 270 a shown in FIG.25 comprises a body 272 a, and a reservoir 274 a containing a coatingcomposition 276 a which comprises, for example, any of the coatingcompositions described elsewhere herein. The applicator cartridge 270 afurther comprises an applicator device 278 a which may have means (notshown) for enabling it to be pushed upwardly into the body 272 a whenthe applicator device 278 a is pressed against a surface such as ananalytic substrate as described elsewhere herein. The applicatorcartridge 270 a further comprises a flexible wall 280 a positionedwithin the body 272 a between the reservoir 274 a and the applicatordevice 278 a for containing the coating composition 276 a within thereservoir 274 a.

The applicator cartridge 270 a further comprises a peristalticpumping/dispensing system 282 a within the body 272 a and which ispositioned partially within the reservoir 274 a and a space 284 abetween the flexible wall 280 a and the applicator device 278 a. Theperistaltic pumping/dispensing system 282 a has a first one way valve286 a and an inlet 288 a, a second one way valve 290 a and a pumpingtube 292 a operatively connected between the first one way valve 286 aand the second one way valve 290 a for receiving a quantity of thecoating composition 276 a from the one way valve 286 a. A dispensingtube 294 a having one or more dispensing ports 296 a exits the secondone way valve 290 a for delivering the quantity of coating composition276 a from the pumping tube 292 a onto the applicator device 278 a whenthe peristaltic pumping/dispensing system 282 a is activated. Theperistaltic pumping/dispensing system 282 a is activated when a rotorsystem 302 comprising one or more cams 304 is electrically activatedwhich causes pressure to be exerted on a portion of the pumping tube 292a thereby comprising the pumping tube 292 a, thereby forcing coatingcomposition therein through the second one way valve 290 a into thedispensing tube 294 a and therefrom through the dispensing ports 296 aand into the applicator device 278 a, which then spreads the coatingcomposition 276 a onto the analytic substrate as described elsewhereherein. When pressure on the pumping tube 292 a is removed, the pumpingtube 292 a is decompressed which causes a new portion of coatingcomposition 276 a to be drawn through the inlet 288 a into the first oneway valve 286 a and therefrom into the pumping tube 292 a. The processcan then be repeated.

Such rotor systems 302 for use in peristaltic pump systems are wellknown by those of ordinary skill in the art. The self-containedperistaltic pumping/dispensing system 282 a is able to dispense veryprecise quantities of the coating composition 276 a by varying theelectrical impulses received thereby. Not only the quantities can beregulated, but also the duration and intervals of the pumping action.The applicator device 278 a may further comprise sensors 306 fordetecting moisture within the applicator device 278 a for monitoring the“wetness” thereof. The peristaltic pumping/dispensing system 282 a mayact automatically based on information from the sensors 306 to deliverquantities of the coating composition 276 a to the applicator device 278a to maintain desired levels of “wetness” during or between use of theapplicator cartridge 270 a. The applicator cartridge 270 a (or any otherapplicator cartridge described or contemplated herein) may furthercomprise one or more internal sensors 308 for measuring and detectinglevels of the coating composition 276 a within the reservoir 274 a fordetermining when the applicator cartridge 270 a is nearly empty, orapproaching a predetermined degree of emptiness, wherein the user maydispose of and replace the applicator cartridge 270 a or refill it incertain embodiments. Alternate pumping/dispensing systems contemplatedfor use in the applicator cartridges of the present invention includethose shown in U.S. Pat. Nos. 6,991,214, and 7,011,397, and U.S. Patentscited therein, all of which are hereby expressly incorporated herein byreference in their entireties.

In an alternate embodiment of the invention, an applicator cartridge 270b shown in FIG. 26 comprises a body 272 b, and a reservoir 274 bcontaining a coating composition 276 b which comprises, for example, anyof the coating compositions described elsewhere herein. The applicatorcartridge 270 b further comprises an applicator device 278 b which mayhave slide means (not shown) for enabling it to be pushed upwardly intothe body 272 b when the applicator device 278 b is pressed against asurface such as an analytic substrate as described elsewhere herein. Theapplicator cartridge 270 b further comprises a flexible wall 280 bpositioned within the body 272 b between the reservoir 274 b and theapplicator device 278 b for containing the coating composition 276 bwithin the reservoir 274 b.

The applicator cartridge 270 b further comprises a peristalticpumping/dispensing system 282 b within the body 272 b and which ispositioned partially within the reservoir 274 b and a space 284 bbetween the flexible wall 280 b and the applicator device 278 b. Theperistaltic pumping/dispensing system 282 b has a first one way valve286 b and an inlet 288 b, a second one way valve 290 b and a pumpingtube 292 b operatively connected between the first one way valve 286 band the second one way valve 290 b for receiving a quantity of thecoating composition 276 b from the one way valve 286 b. A dispensingtube 294 b having one or more dispensing ports 296 b exits the secondone way valve 290 b for delivering the quantity of coating composition276 from the pumping tube 292 b onto the applicator device 278 b whenthe peristaltic pumping/dispensing system 282 b is activated. Theperistaltic pumping/dispensing system 282 b is activated when a pistonrod 310 within the body 272 is pushed downwardly via action of a steppermotor 312 against the pumping tube 292 b which in turn is pressedagainst a rigid plate 314 which thereby compresses the pumping tube 292b, thereby forcing coating composition therein through the second oneway valve 290 b into the dispensing tube 294 b and therefrom through thedispensing ports 296 b and into the applicator device 278 b, which thenspreads the coating composition 276 b onto the analytic substrate asdescribed elsewhere herein. When pressure from the piston rod 310 isreleased, the pumping tube 292 b is decompressed which causes a newportion of coating composition 276 b to be drawn through the inlet 288 binto the first one way valve 286 b and therefrom into the pumping tube292 b. The process can then be repeated.

Shown in FIG. 27 applicator cartridge is an alternate embodiment of thepresent invention. An applicator system 320 comprises an applicatorcartridge 322 having a peristaltic pumping/dispensing system 324 and anapplicator device 326 which is remote from the applicator cartridge 324and is connected thereto via a pumping tube 328 which has dispensingports 330 for dispensing a quantity of a coating composition 332 ontothe applicator device 326. The peristaltic pumping/dispensing system 324is constructed and operates in a manner similar to the peristalticpumping/dispensing system 282 a or 282 b of applicator cartridges 270 aand 270 b, respectively. Alternatively, the applicator system 320 maycomprise an applicator stylus 334 in lieu of, or in addition to, theapplicator device 326.

Shown in FIG. 28 is an alternate embodiment of an applicator cartridgeof the present invention. An applicator system 320 a comprises anapplicator cartridge 322 a and a peristaltic pumping/dispensing system324 a positioned outside of applicator cartridge 322 a and an applicatordevice 326 a which is remote from the applicator cartridge 324 a and isconnected thereto via a pumping tube 328 a which has dispensing parts330 a for dispensing a quantity of a coating composition 332 a onto theapplicator device 326 a. The peristaltic pumping/dispensing system 324 ais constructed and operates in a manner similar to the peristalticpumping/dispensing system 282 a or 282 b of applicator cartridges 270 aand 270 b, respectively. Alternatively, the applicator system 320 a maycomprise an applicator stylus 334 a in lieu of, or in addition to, theapplicator device 326 a.

Shown in FIG. 29 is an alternate version of a movable applicatorcartridge holder of the present invention represented by the generalreference numeral 340. The movable applicator cartridge holder 340supports three separate applicator cartridges 342, 344, and 346, andother embodiments, may hold two, four, or more such applicatorcartridges. The applicator cartridges 342, 344, and 346 may be any ofthe applicator cartridges described or contemplated elsewhere herein.The movable applicator cartridge holder 340 can be used as a componentof any of the analytic substrate coating apparatuses described orcontemplated herein.

The movable applicator cartridge holder 340 can have multiple applicatorcartridges and applicators to treat a single analytic substrate withseveral different coating solutions thereon or to treat a singleanalytic substrate with only one coating solution substrate and mayfurther have the ability to treat multiple analytic substrates at thesame time (simultaneously) with either one or several coatingcompositions depending on the desired coating or coatings for eachanalytic substrate. Therefore, one analytic substrate could be treatedwith one type of coating composition while another analytic substratecould be treated with another type of coating composition or the samecoating composition simultaneously.

The analytic substrate is preferably used by the user immediately (e.g.,within 60 seconds, or within 10 minutes, or within 30 minutes, or within60 minutes, or within 180 minutes, or within 480 minutes, or within 720minutes) after the analytic substrate is coated by one of the analyticsubstrate coating apparatuses as described elsewhere herein, that is,preferably there is no rinsing means or rinsing step in the processbefore a biological specimen is finally applied to the coated analyticsubstrate. Thus, the method of using the analytic substrate ispreferably absent a step of rinsing the analytic substrate in a solventafter the coating is applied to the analytic substrate and before thebiological specimen is applied to the coated analytic substrate.

In a preferred embodiment relating to attachment of paraffin-embeddedtissue sections to an analytic substrate, the analytic substrate iscoated in a method as described elsewhere herein and within seconds orminutes the paraffin-embedded tissue section is floated from thehistologic water bath onto the coated portion of the analytic substrate.The analytic substrate with the biological specimen attached isimmediately placed onto a 50° C.-100° C. hotplate or in a 50° C.-100° C.oven for 5-60 minutes, and preferably for 10-20 minutes at 60° C.Heating can also be performed in a microwave oven.

The process of heating the analytic substrate after the tissue has beenattached is well known in the art and variations of this heating stepare known and are used with the laboratory produced and commerciallyproduced coated analytic substrates. This known heating step dries thewater from underneath the tissue section and melts the paraffin causingthe tissue section to contact the glass.

The unanticipated benefit realized by producing a coated analyticsubstrate using the present invention is that this heating stepincreases the chemical binding nature (chemical reactivity) of thefreshly coated primer or coupling agent (e.g., silane, siloxane) to theglass analytic substrate surface (causing siloxane bond formation) andthe chemical binding of the tissue to the functional group of the primeror coupling agent, thereby producing a superior link of the tissue tothe glass surface.

This heating step used with paraffin embedded tissue sections causesmelting of the paraffin (e.g., at temperatures over 60° C.) wherein themelted paraffin rises to the top of the trapped water layer under thetissue section exposing the tissue to the functional groups of thecoating. The heat, along with the movement of the paraffin away from theunderneath of the tissue, and the possible production of alcohol,induces the tissue section to lay flat against the glass surface andfacilitates binding of it to the functional groups of the coating forsuperior attachment of the tissue section to the glass surface. Sincethe present invention produces a superior coated analytic substrate, thebenefits of increased adhesion to the biological specimen to theanalytic substrate surface also occur under conditions of air drying,mild heat (e.g., less than 50° C.), microwave heating, or partial airdrying then heating, for example.

This outcome due to heating, relating to the chemical nature of thecoating, is absent when prior art coated analytic substrates are usedbecause the coating in prior art analytic substrates was prepared days,months, or years before the tissue is attached to the analyticsubstrate. The minimal siloxane bonds formed on the prior art analyticsubstrates are completed and finalized before attachment of the tissuesection rather than during or after. It is believed that furthersiloxane bonding does not occur after the tissue section is placed onthe analytic substrate when the prior art analytic substrates are used.Therefore, in the prior art analytic substrates, the heat serves nobenefit to the chemical binding nature of the coating, only to dry thewater from underneath the tissue section. This heating step can be usedto increase the chemical binding nature of the coating of the presentinvention when attaching other biological specimens such as aredescribed elsewhere herein.

In a further embodiment of the present invention, the coating apparatusis used to apply an overcoat upon the specimen on the analytic substrateafter all testing has been performed and the specimen on the analyticsubstrate requires a permanent mounting to seal the specimen for viewingunder a microscope or other viewing source (i.e., visual, computer aidedscanning, etc.) and subsequent archival storage. Previously, glass orplastic cover slips have been disposed over the specimen with mountingmedia to permanently seal the biological specimen thereon. The presentinvention eliminates the need for this cumbersome coverslippingtechnique.

In the present embodiment, after the final step in the processing of thespecimen, the functional side of the analytic substrate having thebiological specimen thereon is spray-coated, or coated via theapplicator device by wiping, to apply an overcoat onto the functionalside of the analytic substrate. Once the overcoat is dry the specimenbecomes sealed permanently. The overcoat can have substantially the samethickness as a normal glass or plastic cover slip. Preferably therefractive index of the overcoat is suitable for viewing under lightmicroscopy, electron microscopy, computer aided scanning, and human eyeviewing like a regular glass or plastic cover slip. The overcoat maycomprise, for example a coating solution comprising a liquifiedthermoplastic polymer or a coverslip mounting medium such as the mediumknown commercially as PERMOUNT.

In a preferred embodiment, the overcoat is applied to the analyticsubstrate by an applicator cartridge which sprays the coating materialon the analytic substrate via a non-wiping applicator (e.g., perforatedmetal spray-type applicator such as is known by one of ordinary skill inthe art) which delivers fine micro droplets of coating material ontoanalytic substrate to minimize overspray and wastage. The non-wipingapplicator device can move over the functional side of the analyticsubstrate one or several times to deposit the overcoat to attain thethicknesses of commercial cover glass and/or plastic cover slips. Thethickness of the overcoat which can be applied by the apparatus ispreferably from 0.001 mm to 5 mm.

In one embodiment the overcoat is applied by an applicator cartridge(not shown) which is similar to any one of applicator cartridges 14, 70,184, 270, 270 a, 270 b, 320, and 320 a shown herein except that theapplicator cartridge for forming the overcoat lacks an applicator deviceable to wipe the overcoat coating composition and instead is equippedwith a spray nozzle or other spraying or atomizing device for applyingthe overcoat coating composition. In an alternate embodiment applicatorcartridges 14, 70, 270, 270 a, 270 b, 320, and 320 a may be constructedso the applicator devices thereon can be retracted so that the coatingcomposition can be applied only by a spray-type device and not wipedthereon.

In summary, the invention contemplates a portable, point-of-useapparatus and method of its use as described herein. The invention, in apreferred embodiment, contemplates using this apparatus to coat analyticsubstrates with a coating composition having adhesive properties,hydrophobic properties, hydrophilic properties, and/or ionic properties(cationic, anionic, and neutral) by using a wiping step, or a non-wiping(e.g., spraying) step, and is absent a rinsing step. The analyticsubstrates are then used “on site”, i.e., in the same laboratory orfacility in which the coated analytic substrates are produced.Optionally, the user can employ an additional step to apply an overcoatof a coating material such as a liquified thermoplastic polymer (or acover slipping medium such as the commercially-available PERMOUNT) topermanently bond and seal a specimen on the analytic substrate.

In a preferred embodiment, the replaceable or refillable applicatorcartridges and/or reservoirs contemplated herein have machine-readableinsignias, numbers, identifiers, codes, barcodes, symbols, and any otherreadable patterns known in the art of automated identification ofproducts by automated readable instrumentation (herein also known as“machine readable patterns”) located on their outside surfaces (notshown). These “machine readable patterns” located on the surfaces of theapplicator cartridges and/or reservoirs can be read or scanned by themicroprocessor of the present apparatus, or by other means foridentification and validation of empty, refilled, and/or newly replacedapplicator cartridges and/or reservoirs. This scanned informationcontained in the readable patterns can identify lot numbers, expirationdates, composition of the coating, manufacture date, remaining coatingdispenses, dispenses used in an automated run, and other pertinentinformation relating to the applicator cartridges and/or reservoirs.This information can be stored in the database of the microprocessor forretrieval by the technician. An applicator cartridge and/or reservoircan be scanned at any time. The microprocessor can scan the readablepatterns when a new applicator cartridge and/or reservoir is beingplaced into the coating apparatus, when an applicator cartridge and/orreservoir is empty, or when an empty applicator cartridge and/orreservoir needs to be refilled and is replaced by notifying thetechnician of the status of the applicator cartridge and/or reservoir atall times before, during, and after a coating process. The readablepatterns can also be used to identify which applicator cartridge and/orreservoir is to be used to dispense a coating onto a particular analyticsubstrate during a procedure, therefore eliminating human error relatingto the coating processes when working with several different types ofcoating solutions and/or patterns (borders and/or coating locations)when utilizing this processes of automated coating of analyticsubstrates as related to the present invention.

The present invention is not to be limited in scope by the specificembodiments described herein, since such embodiments are intended as butsingle illustrations of one aspect of the invention and any functionallyequivalent embodiments are within the scope of this invention. Indeed,various modifications of the methods of the invention in addition tothose shown and described herein will become apparent to those skilledin the art form the foregoing description.

All U.S. Patents, patent applications, and cited references are herebyexpressly incorporated herein by reference in their entireties.

What is claimed is:
 1. A method, comprising: applying a coating on ananalytic substrate to form a coated surface on at least a portion of theanalytic substrate, wherein the coating comprises organofunctionalsilane; applying a biological specimen to the coated surface of theanalytic substrate; and producing a hydrolytic area between thebiological specimen and the analytic substrate wherein in situhydrolysis of the coating is initiated by water between the biologicalspecimen and the analytic substrate to cause the biological specimen tobind to the analytic substrate.
 2. The method of claim 1, wherein whenthe biological specimen is applied to the coated analytic substrate, theorganofunctional silane in the coating becomes bound by in situhydrolysis and condensation of at least one reactive silyl group thereofto the analytic substrate, and wherein the biological specimen binds toat least one functional group of the organofunctional silane whereby thebiological specimen becomes bound to the analytic substrate.
 3. Themethod of claim 1, wherein the analytic substrate is a microscope slide.